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4. TITLE AND SUBTITLE The Role of IQGAP1 in Breast Carcinoma
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6. AUTHOR(S) Dr. Colin D. White, PhD
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Brigham and Women’s Hospital Inc
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Boston Massachusetts 02115
9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Material Command Fort Detrick 11. SPONSOR/MONITOR’S REPORT Maryland NUMBER(S) 21702 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT HER2 is overexpressed in ~25% of breast carcinomas. Overexpression of HER2 is an adverse prognostic feature and correlates with shorter disease-free and overall survival. HER2(+) breast cancer is treated with trastuzumab but many patients do not respond. Of those who do, most become refractory to therapy and progress to metastatic disease. An insight into the molecular mechanisms underlying HER2 signaling and trastuzumab resistance is essential to reduce breast cancer morbidity and mortality. IQGAP1 is a ubiquitously expressed scaffold protein that contains multiple protein interaction domains. Through interaction with its binding partners, IQGAP1 integrates diverse signaling pathways, several of which are relevant to breast tumorigenesis. The purpose of this proposal is to elucidate the function of selected IQGAP1 binding interactions in breast neoplasia. During Year 1 of this fellowship, we have shown that IQGAP1 interacts with HER2 in vitro and in a normal cellular milieu. Furthermore, IQGAP1 and HER2 co-immunoprecipitate from SkBR3 cells. The remainder of this award will be spent evaluating the functional consequences of this interaction on HER2 signaling and trastuzumab resistance. Elucidation of the molecular mechanism underpinning this interaction could potentially lead to the development of novel and specific therapeutic agents for the treatment of patients with HER2(+) breast cancer. 15. SUBJECT TERMS Breast Cancer, Drug Resistance, HER2, IQGAP1, Trastuzumab
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The IQGAP family comprises three proteins in humans. The best characterized is IQGAP1, which par-ticipates in protein–protein interactions and integrates diverse signaling pathways. IQGAP2 andIQGAP3 harbor all the domains identified in IQGAP1, but their biological roles are poorly defined.Proteins that bind IQGAP1 include Cdc42 and Rac1, E-cadherin, b-catenin, calmodulin and compo-nents of the mitogen-activated protein kinase pathway, all of which are involved in cancer. Here,we summarize the biological functions of IQGAPs that may contribute to neoplasia. Additionally,we review published data which implicate IQGAPs in cancer and tumorigenesis. The cumulative evi-dence suggests IQGAP1 is an oncogene while IQGAP2 may be a tumor suppressor.! 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
IQGAPs comprise a class of multidomain proteins, which arepresent in diverse organisms ranging from yeast and Caenorhabdi-tis elegans to Xenopus laevis and mammals [1]. There are threeIQGAPs in humans (Fig. 1). The first to be described was the190-kDa protein IQGAP1, which was cloned in 1994 [2]. IQGAP2,which is 62% identical to IQGAP1, was identified 2 years later [3]and IQGAP3 was isolated in 2007 [4]. The vast majority (>85%) ofthe published literature focuses on IQGAP1. Less is known aboutIQGAP2 (20 primary papers in PubMed [http://www.ncbi.nlm.nih.gov/pubmed] at the time of writing) and there are only two pri-mary papers on IQGAP3. The cytoskeletal [5–8] and cellular signal-ing [1,9] functions of IQGAP1 have been extensively reviewed inthe last few years. Here, we briefly compare the characteristics ofIQGAP1, IQGAP2 and IQGAP3, then focus on published data thataddress their involvement in neoplasia.
2. Comparison of human IQGAP proteins
The IQGAP proteins share a similar domain structure and haveconsiderable sequence homology (Fig. 1). These domains mediatethe association of IQGAPs with a diverse spectrum of proteins[9]. Binding to IQGAP1 modulates the function of the interactingproteins, resulting in the alteration of multiple cellular behaviors[5,7,9]. Despite limited information on IQGAP2 and IQGAP3, it isapparent that they differ from IQGAP1 in several respects (includ-ing tissue distribution, subcellular localization and interaction withbinding proteins). These distinctions may account for some of thefunctional differences among the three IQGAPs that are beginningto emerge.
The tissue distribution of the IQGAP proteins varies consider-ably. IQGAP1 has ubiquitous expression [2]. IQGAP2 is found pre-dominantly in liver, but can be detected in prostate, kidney,thyroid, stomach, testis, platelets and salivary glands [3,4,10,11],while IQGAP3 is reported to be present in brain, lung, testis, smallintestine and colon [4,12].
IQGAPs exhibit both similarities and differences in their subcel-lular localization. In human epithelial cells in culture, endogenousIQGAP1 is distributed throughout the cytoplasm and accumulatesat cell–cell junctions where it co-localizes with E-cadherin [13].In quiescent human platelets IQGAP2 demonstrates diffuse cyto-plasmic staining [10]. When platelets are activated, IQGAP2 isfound predominantly in filopodia, with less prominent staining in
0014-5793/$36.00 ! 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.doi:10.1016/j.febslet.2009.05.007
the cell body. By contrast, IQGAP2 is predominantly localized in thenucleus and at sites of cell–cell contact in isolated rabbit gastricglands in primary culture [14]. In this study, IQGAP1 was observedto be targeted predominantly to the cortex of chief and mucousneck cells. These findings contradict those of an earlier publicationwhere IQGAP1 and IQGAP2 were localized to the basolateral andapical membranes, respectively, in rabbit gastric parietal cells[15]. The localization of IQGAP3 in human cells has not been de-scribed. In PC12 rat phaeochromocytoma cells IQGAP3 is diffuselydistributed in the cytoplasm [4], while in cultured Eph4 mouse epi-thelial cells it is found at cell–cell junctions [12]. IQGAP1 and IQ-GAP3 have similar distribution in the cell bodies, distal parts ofaxons and axon growth cones of rat embryo hippocampal neurons[4]. Interestingly, IQGAP3 expression is reported to be confined toproliferating cells [12]. Additional studies are necessary to clearlydelineate the subcellular distribution of IQGAP2 and IQGAP3 in hu-man tissue.
3. IQGAP binding proteins
IQGAP1 binds numerous proteins [9]. Much less is known aboutthe binding partners of IQGAP2 and IQGAP3. Nevertheless, suffi-cient information is now available to permit one to begin to teaseout differences. IQGAP1 binds to GTP-Cdc42 and GTP-Rac1 withsubstantially higher affinity than to the inactive, GDP-bound formof the GTPases [16,17]. Similarly, the interaction of IQGAP3 withRac1 and Cdc42 appears to be GTP-dependent [4]. In contrast,although not observed in all cases [15], IQGAP2 has been reportedto interact with both the GDP- and the GTP-bound forms [3,18].Another protein that may bind differentially to IQGAPs is Ras. Nointeraction between H-Ras and IQGAP1 [16,18,19] or IQGAP2[3,18] has been detected (IQGAP1 was identified in a complex withM-Ras [20], but direct binding has not been demonstrated). For IQ-GAP3, the evidence is contradictory. One group reported that Rasbinds to IQGAP3 [12], while other investigators failed to observean interaction between the two proteins [4]. Further work is re-quired to reconcile these discrepant findings and provide detailedanalysis of the binding partners of IQGAP2 and IQGAP3.
It is important to emphasize that, despite the presence of adomain with sequence similarity to RasGAPs, none of the IQGAPshave GTPase-activating protein (GAP) activity. As mentioned,IQGAP proteins bind to the Rho family GTPases Rac1 and Cdc42.These proteins act as molecular switches by cycling between‘‘on” GTP-bound and ‘‘off” GDP-bound states [21]. Interaction witha GAP accelerates GTP hydrolysis leading to inactivation. Byassociating with GTP-bound Rac1 and Cdc42, IQGAP1 and IQGAP2inhibit the intrinsic rate of GTP hydrolysis and thus stabilize theactive GTP-bound state [3,16,22]. Consistent with these in vitrofindings, overexpression of IQGAP1 in MCF-7 malignant humanbreast epithelial cells increases the amount of active Cdc42 andRac1 [22,23].
In addition to regulating Rac1 and Cdc42 signaling, recent workhas shown that IQGAPs, and IQGAP1 in particular, modulate manydifferent signaling pathways and cellular functions [9], includingmitogen-activated protein kinase (MAPK) signaling, Ca2+/calmodu-lin signaling, cell–cell adhesion, b-catenin-mediated transcriptionand microbial invasion [9,24,25]. Ca2+ and calmodulin appear tobe of great importance to the function of IQGAP1. Calmodulinbinds to IQGAP1 in a Ca2+-regulated manner [16,17], and associa-tion with calmodulin inhibits the ability of IQGAP1 to interact withevery other binding partner studied to date [1,9].
A finding with particular relevance to the topic of this review isthat many IQGAP1 binding partners have well-defined roles intumorigenesis (Table 1). These proteins include the well-describedoncogenes b-catenin and Src, the tumor suppressor E-cadherin, theRho GTPases Cdc42 and Rac1, and components of the MAPK cas-cade. These observations, in conjunction with the ability of IQGAP1to modulate fundamental cellular functions, have led to consider-able attention being directed towards IQGAP1 in the field of cancerbiology. Work from several laboratories suggests that IQGAP1 is anoncogene that promotes both tumorigenesis and metastasis and, toa more limited extent, implies that it may be a useful tumor mar-ker. IQGAP2, in contrast, appears to have the opposite effect andmay act as a tumor suppressor. Little can be inferred regardingthe possible role of IQGAP3 in neoplasia. Here, we review the avail-able evidence for the involvement of IQGAPs in the regulation ofsignaling pathways and cellular functions known to be involvedin neoplastic transformation and/or tumor progression. Addition-ally, we discuss how, by virtue of their cellular expression and/orlocalization, IQGAPs are directly implicated in cancer. While mostof the data presented are germane only to IQGAP1, we also discussthe possible roles of IQGAP2 and IQGAP3 where published evi-dence is available.
4. IQGAP functions with potential relevance to cancer
IQGAP1 regulates many different cellular processes, and chang-ing intracellular IQGAP1 expression or function can alter theseactivities [1,5,7,9]. Therefore, IQGAP1 appears to be important fornormal cellular function and homeostasis. The contribution ofsome of these cellular activities to different stages of cancer pro-gression provides a clear link between IQGAP1 and cancer. Selectedcell functions, and how the IQGAPs control them, will be described.
4.1. MAPK signaling
The MAPK pathway, which modulates multiple cellular pro-cesses, such as differentiation, proliferation and migration, isderegulated in neoplasia [26,27]. For example, mutations in Ras[28] or B-Raf [29,30] are highly prevalent in neoplasms. Activatingmutations in Ras have been reported in over 15% of all human tu-mors and in pancreatic carcinoma this frequency may be as high as
Fig. 1. A schematic diagram of human IQGAP proteins. Domain structure (adapted from SMART and Pfam databases) and percentage amino acid identity of human IQGAP1,IQGAP2 and IQGAP3 are shown. CHD, calponin homology domain; WW, poly-proline protein–protein domain; IQ, IQ domain (with four IQ motifs); GRD, RasGAP-relateddomain; RasGAP_C, carboxy-terminal sequence found in members of the IQGAP family.
1818 C.D. White et al. / FEBS Letters 583 (2009) 1817–1824
90% [28]. In addition, increased extracellular signal-regulated ki-nase (ERK) phosphorylation and expression has been found in pan-creatic cancer [31], and enhanced ERK phosphorylation correlateswith tumor progression in prostate cancer [32]. Increased MAPKkinase (MEK) phosphorylation has been identified in colon cancer[33] and in 74% of myeloblasts in acute myelogenous leukemia[34].
IQGAP1 is a MAPK scaffold, which binds directly to and modu-lates the functions of B-Raf [35], MEK [36] and ERK [37]. IQGAP1 isrequired for activation of B-Raf by epidermal growth factor (EGF)[35]. Similarly, IQGAP1 regulates the activation of MEK and ERKin response to both EGF [36,37] and CD44 [38]. Thus, IQGAP1 is re-quired for efficient propagation of the MAPK cascade.
EGF differentially modulates the association of components ofthe MAPK pathway with IQGAP1. Knockout of IQGAP1 from cellsrenders B-Raf insensitive to EGF stimulation, while B-Raf associ-ated with IQGAP1 has a much higher kinase activity comparedwith free B-Raf [35]. It is unclear whether interacting with IQGAP1enhances activation of B-Raf by EGF, or whether IQGAP1 preferen-tially associates with B-Raf that has already been activated. Inter-estingly, while ERK binds constitutively to IQGAP1 and thebinding is not sensitive to EGF, the interaction between IQGAP1and MEK1 increases, while that with MEK2 decreases, followingEGF treatment [36]. This raises the possibility that IQGAP1 prefer-entially activates the MEK1 signaling pathway. It has been sug-gested that MEK1 promotes proliferation, while MEK2 promotesdifferentiation [39]. Scaffold proteins serve as signaling nodes,influencing signal intensity, time course and the specific cellularresponse to an extracellular cue [40–42]. Thus, the scaffold func-tions of IQGAP1 may modulate the cellular response to activationof MAPK signaling, enhancing proliferation and reducing differen-tiation. These changes could contribute to neoplasia.
Analogous to IQGAP1, siRNA-mediated knockdown of IQGAP3suppresses ERK phosphorylation and significantly reduces prolifer-ation of Eph4 mammary epithelial cells [12]. Moreover, exogenousexpression of IQGAP3 induces a proliferative response, which isblocked by the ERK inhibitor U0126. Thus, it appears that IQ-GAP3-induced ERK activation may have a role in the regulationof cellular proliferation.
4.2. b-Catenin-mediated transcription
b-Catenin, the central molecule in theWnt signaling pathway, isintegral to the control of cellular proliferation and cell–cell adhe-sion, both of which are deregulated in malignancy [43,44]. Underunstimulated conditions, b-catenin is held in a complex with ade-
nomatous polyposis coli (APC) and axin, and is targeted for degra-dation by casein kinase 1 (CK1) and glycogen synthase kinase-3b(GSK-3b). In response to Wnt stimulation, CK1 and GSK-3b areinhibited, and b-catenin accumulates in the cytoplasm from whereit translocates to the nucleus. Here, it promotes gene transcriptionby binding to the T cell factor/lymphoid enhancer factor family oftranscription factors.
IQGAP1 binds directly to b-catenin. Overexpression of IQGAP1enhances b-catenin nuclear localization and b-catenin-dependenttranscription in SW480 colon carcinoma and human bronchial epi-thelial cells [45,46]. Furthermore, targeted disruption of the mur-ine Iqgap2 gene results in increased expression of IQGAP1 in thecytoplasm of hepatocytes, with a concomitant increase in cytoplas-mic b-catenin, b-catenin activation and expression of cyclin D1 (anuclear target of the Wnt/b-catenin pathway) [47]. Taken together,these findings suggest that IQGAP1 is an important regulator of b-catenin function.
4.3. Cellular proliferation
Uncontrolled cellular proliferation is a fundamental characteris-tic of neoplastic transformation. Recent studies have shown thatIQGAP proteins are important regulators of the proliferative re-sponse. Overexpression of IQGAP1 increases proliferation of MCF-7 cells, an effect dependent, at least in part, on increased activeRac1 and Cdc42 [23]. Similarly, IQGAP1 is required for vascularendothelial-derived growth factor (VEGF)-stimulated proliferationas knockdown of IQGAP1 with siRNA abrogates proliferation of hu-man umbilical vein [48] and aortic [49] endothelial cells. Theseobservations suggest that the IQGAP1 expression level directly dic-tates the rate of cellular proliferation. Indeed, siRNA-induced IQ-GAP1 knockdown significantly reduces VEGF-stimulatedangiogenesis in vivo [49]. Furthermore, quercetin, an anti-oxida-tive flavonoid which is known to have strong anti-proliferativeproperties [50], decreases IQGAP1 expression in HepG2 humanhepatocellular carcinoma (HCC) cells [51]. Interestingly, researchin the small intestine has shown that IQGAP3, but not IQGAP1 orIQGAP2, is exclusively expressed in proliferating cells [12].
4.4. Cell–cell adhesion
Decreased tumor cell adherence at the primary site, increasedproteolytic degradation of surrounding tissue and enhanced cellmotility are required for cancer cells to metastasize. Loss of cell–cell adhesion occurs as a result of reduced E-cadherin function,but the precise molecular mechanisms underlying this effect are
Table 1IQGAP1 binding proteins with identified roles in cancer.
Bindingpartner
Effect of IQGAP1 on target protein Functional consequence of interaction Potential relevance to cancer References
cell–cell adhesionDisrupts cell–cell adhesion Transformation and metastasis [45,53,95]
B-Raf MAPK scaffold; increases B-Raf activity Enhances proliferation and angiogenesis Transformation [35,49]Calmodulin Prevents interaction of IQGAP1 with other binding
proteinsRegulates IQGAP1 functions Proliferation and metastasis [16,17,96]
CD44 Links hyaluronan to actin cytoskeleton Promotes migration Metastasis [38]Cdc42/Rac1 Stabilizes active form Promotes proliferation, cell motility and
C.D. White et al. / FEBS Letters 583 (2009) 1817–1824 1819
poorly understood [52]. E-cadherin mediates intercellular adhe-sion through homophilic associations with the extracellular do-mains of E-cadherin on a neighboring cell. Importantly, IQGAP1binds directly to E-cadherin [13,53] and overexpression of IQGAP1reduces E-cadherin-mediated adhesion [53]. Similarly, transloca-tion of IQGAP1 to cell–cell junctions attenuates E-cadherin func-tion [13].
While no published studies have investigated the possible roleof mammalian IQGAP2 or IQGAP3 in cell–cell adhesion, a X. laevisIQGAP2 homolog (XIQGAP2) localizes at cell–cell junctions in bothcultured Xenopus cells and embryos [54]. Suppression of XIQGAP2expression by microinjection of morpholino antisense oligonucleo-tides results in ectodermal lesions in mid-neurula stage embryosdue to loss of cell–cell adhesion [55]. These findings suggest thatXIQGAP2 expression positively regulates cell–cell adhesion duringearly development. It remains to be determined whether IQGAP2contributes to the maintenance of cell–cell adhesion in mammals.
4.5. Exocytosis
Tumor cell invasion across tissue boundaries is dependent on thecapacity of neoplastic cells to breach the basement membrane andremodel the extracellular matrix (ECM), events which commonlyoccur by proteolytic cleavage bymatrixmetalloproteinases (MMPs)[56]. ActiveMMPs are delivered to the sites of contact between inva-sive tumor cells and the ECM via dynamic cellular protrusionsknown as invadopodia [57,58]. MMP accumulation at invadopodiais thought to rely on vesicle exocytosis which, in turn, depends onthe successful targeting and tethering of vesicles to appropriate siteson the cell membrane. Here, the exocyst, a multiprotein complexconsisting of eight subunits including Sec3, Sec8 and Exo70 [59], isbelieved to play a pivotal role. Importantly, IQGAP1 binds Sec3,Sec8 and Exo 70 [60,61], implicating IQGAP1 in the regulation ofexocytosis. Moreover, interaction between IQGAP1 and the exocystwas shown to benecessary for invadopodia activity inMDA-MB-231cells [60]. Interestingly, silencing IQGAP1 inhibits the invasion ofovarian carcinoma HO-8910PM cells in vitro [62] and MCF-7 cellsin vitro and in vivo [23,63]. It is therefore tempting to speculate thatIQGAP1 mediates invasion, at least in part, by regulating exocytosisand subsequent degradation of the ECM.
4.6. Cell migration
Most cancer deaths are caused by metastatic disease. The mech-anism by which metastases develop remains to be fully elucidated,
but it is agreed that cells must have both invasive and migratoryproperties [64]. Many factors which are known to increase cellmigration in vitro have been shown to promote metastasisin vivo [64]. IQGAP1 was originally characterized as regulator ofRac1/Cdc42 signaling and actin dynamics [1,5–7], and much ofthe early work on IQGAP1 focused on its role in regulating thecytoskeleton. Consequently, IQGAP1 was observed to be an impor-tant modulator of cell migration [63,65–67]. IQGAP1 cross-linksactin filaments [5,68], and localizes to the leading edge of migrat-ing cells [63,66]. Increasing IQGAP1 expression in cells increasesthe amount of active Cdc42 and promotes cell migration [63],although other IQGAP1 binding partners, including actin, calmodu-lin [69], and APC [66], are also likely to contribute to this effect.siRNA-induced knockdown of IQGAP1 reduces the migration ofseveral human cell lines, such as MCF-7 [63] and U87MG humanglioblastoma cells [70]. In agreement with these studies, IQGAP1is required for the induction of cell migration by fibroblast growthfactor (FGF), VEGF and hyaluronan [38,48,71].
5. Role of IQGAP proteins in cancer
The work outlined above implicates the IQGAP proteins, partic-ularly IQGAP1, in neoplasia by virtue of their cellular functions.Nevertheless, it is important to note that much of these data wereobtained from cultured cell lines, and a critical reader may arguethat their relevance to cancer is largely circumstantial. In the fol-lowing sections we discuss evidence derived from human neo-plasms and mouse models of cancer, which more directlyidentify the involvement of IQGAP1 and IQGAP2 in neoplastictransformation and metastasis.
5.1. Genetic studies
The level of expression of IQGAP genes and mRNAs are fre-quently altered in neoplasia. IQGAP1 has been proposed to be anoncogene [23]. Consistent with this postulate, comparison of thegenetic profiles of tumors with those of normal tissue, and compar-ison of more aggressive cancers with less aggressive neoplasms, re-veals that the Iqgap1 gene and/or mRNA are overexpressed in allanalyses reported (Table 2). Increased expression of Iqgap1 hasbeen observed in several human neoplasms, including lung [72],colorectal [73] and oligodendroglioma [72]. Analogous observa-tions have been reported in mouse models (Table 2). Iqgap1 isoverexpressed in a genetically engineered mouse that recapitulatesthe stages of human prostate cancer progression [74]. While not
Table 2Changes in Iqgap gene/mRNA expression level in neoplasms.B
Tissue comparison IQGAP Species Method Neoplasm Expression change References
Cancer vs. normal Iqgap1 Human Array Colorectal + [73]Glioma + [97]
RT-PCR Glioma + [97]Lung + [72]
Mouse Array Prostate + [74]Iqgap2 Human Array Colorectal + [78]
Prostate + [79]
Aggressive vs. less aggressive cancerA Iqgap1 Human Array Glioma + [88]a
Mouse Array Medulloblastoma + [98]b
Melanoma + [90]c
Iqgap2 Human Array Prostate ! [77]d
Mouse Array Prostate ! [74]e
Iqgap3 Human Array Colorectal ! [99]f
+, Increased expression; !, decreased expression.A Aggressive vs. less aggressive cancer is defined by a decrease in long-term survivala,f, an increase in the likelihood of metastasisb,c,e or a decline in tumor responsiveness to
hormone therapyd.B Only published studies with primary tissue are included. Data with cultured cell lines have been omitted.
1820 C.D. White et al. / FEBS Letters 583 (2009) 1817–1824
included in Table 2, similar findings have been documented in cul-tured cell lines. For example, the Iqgap1 gene is amplified in HSC39and HSC40A gastric cancer cell lines [75]. This amplification corre-sponds to an increase in both IQGAP1 mRNA and protein, com-pared with normal gastric cell lines, and an accumulation ofIQGAP1 protein at the cell membrane [75].
IQGAP2 has also generated several ‘‘hits” in genetic screenscomparing normal with neoplastic tissue (Table 2). The results,however, are less unequivocal than those for IQGAP1. IQGAP2expression is lost from 5/9 gastric cancer cell lines due to aberrantmethylation of the IQGAP2 promoter [76]. This abnormal methyl-ation was also observed in 47% of primary gastric cancer tissues(compared with 0% in normal tissue), and is significantly associ-ated with tumor invasion and a poor prognosis [76]. The inversecorrelation of Iqgap2 expression with cancer progression suggeststhat IQGAP2 may be a tumor suppressor. This hypothesis is sup-ported by studies which show reduced expression of the Iqgap2gene in hormone-refractory prostate cancer [74,77]. However,other reports contradict these findings as they observe overexpres-sion of Iqgap2 in tissue from cancers of the colon [78] and prostate[79] (Table 2). Thus, while there is evidence to suggest that Iqgap2acts as a tumor suppressor, more thorough investigations arerequired in order to verify this postulate and ascertain whether itpertains only to selected neoplasms.
5.2. IQGAP protein expression and localization
The level of expression of IQGAP proteins is also altered in neo-plasia (Table 3). Compared with normal tissue, IQGAP1 is overex-pressed in colorectal carcinoma [80], breast cancer [23],astrocytoma [81] and squamous cell carcinoma of the head andneck [82]. Furthermore, IQGAP1 expression in aggressive ovarianadenocarcinomas is higher than in adenomas or borderline tumors[62], while a lack of IQGAP1 protein expression is associated with afavorable prognosis in gastric cancer [83]. There is little publishedon IQGAP2 protein expression in neoplasia. Only one paper, pub-lished recently, addresses this topic. In this study, IQGAP2 expres-sion is lost from 61% of human gastric carcinoma tissue, but isdetected in 100% of normal gastric mucosa [76]. This observationis consistent with the postulated role for IQGAP2 as a tumorsuppressor.
In addition to increased expression, the subcellular localizationof IQGAP1 is altered in carcinoma. IQGAP1 is localized at the inva-sive front of more aggressive colorectal [80] and ovarian [84] neo-plasms. In the latter study, a diffuse expression pattern of IQGAP1indicates a high histological grade and clinicopathological stage.IQGAP1 overexpression and diffuse invasion pattern were signifi-
cantly associated with poor prognosis by multivariate analysis[84]. Data from other groups support the premise that peripherallocalization of IQGAP1 is associated with more aggressive tumors.For example, translocation of IQGAP1 from the cytoplasm to thecell membrane correlates with dedifferentiation of gastric carci-noma [85]. IQGAP1 is also implicated in endometrial adenocarci-noma [86]. Compared with normal tissue, in well-differentiatedtumors, IQGAP1 disappears from cell adhesion sites, while E-cad-herin is still present around cell boundaries [86]. However, inpoorly differentiated tumors, IQGAP1 and E-cadherin accumulatein aggregates [86], suggesting that IQGAP1 and E-cadherin functionis disrupted in advanced, poorly differentiated tumors.
The connection between IQGAP1 and E-cadherin is also seen ingastric cancers. In normal epithelium, IQGAP1 and E-cadherin arelocalized to the cell–cell boundary [85]. However, IQGAP1 localizesto the cytoplasm in intestinal-type tumors and to the cell periph-ery in diffuse-type tumors [85]. These results are further supportedby fractionation data showing that in differentiated tumors IQ-GAP1 is present in the soluble fraction and E-cadherin in the insol-uble fraction, but both are insoluble in undifferentiated tumors[85]. Consequently, it is thought that as tumor cells de-differenti-ate, IQGAP1 moves from the cytoplasm to the cell periphery whereit disrupts E-cadherin function [85].
There is strong evidence to suggest that IQGAP1 expression canserve as a biomarker for diagnosis of glioblastomas. In a rat modelof glioma, IQGAP1 expression is restricted to a subpopulation ofnestin-positive amplifying tumor cells in glioblastoma-like tumors,but not oligodendroglioma-like tumors [87]. In human glioblas-toma samples, IQGAP1 is a marker for nestin-positive cancer cells,which appear to represent stem-like cancer progenitors [87]. In astudy to identify biomarkers of aggressive gliomas, IQGAP1 expres-sion, along with insulin-like growth factor binding protein-2,identifies a subgroup of patients with grade III gliomas with poorprognosis [88]. While normal glial tissue does not express IQGAP1,cytoplasmic IQGAP1 staining is seen in 64% of gliomas [88].
Finally, outcome studies in other cancers are also beginning toemerge. For example, increased Iqgap1 expression constitutes partof a genetic signature that significantly predicts the likelihood ofrecurrence of colon cancer [89]. While it remains to be elucidatedif these findings are relevant to other tumors, these data suggestthat IQGAP1 may be useful in evaluation of patient prognosis.
5.3. IQGAP1 in metastasis
Genetic evidence using an in vivo scheme indicates a role forIqgap1 in metastasis. A screen for genes exhibiting altered expres-sion in a mouse model of metastatic melanoma identified Iqgap1
Table 3Changes in IQGAP protein expression level in neoplasms.B
Tissue comparison IQGAP Species Method Neoplasm Expression change References
Cancer vs. normal IQGAP1 Human IHC Colorectal + [80]MS Squamous cell (Head and Neck) + [82]WB Astrocytoma + [81]WB Breast + [23]
IQGAP2 Human IHC Gastric ! [76]Mouse IHC/WB Liver ! [47]
Aggressive vs. less aggressive cancerA IQGAP1 Human IHC Gastric + [83]a
Glioma + [88]b
Lung + [100]c
Ovary + [84]d
IHC/WB Glioma + [87]e
+, Increased expression; !, decreased expression.Abbreviations: IHC, immunohistochemistry; WB, western blot; MS, mass spectrometry.
A Aggressive vs. less aggressive cancer is defined by a decrease in long-term survivala,b,d or an increase in the likelihood of metastasisc,e.B Only published studies with primary tissue are included. Data with cultured cell lines have been omitted.
C.D. White et al. / FEBS Letters 583 (2009) 1817–1824 1821
and its binding proteins calmodulin and ERK as 3 of only 32 genes(from !10500 arrayed genes) that showed a >2.5-fold increase inexpression in metastatic cells [90]. The small number of genesidentified implies that increased expression of IQGAP1 and cal-modulin are likely to be important in metastasis, rather than anindirect result of the altered phenotype.
6. IQGAPs in tumorigenesis
As we have highlighted, accumulating evidence reveals that IQ-GAP1 expression, both RNA and protein, is increased in several hu-man malignancies. IQGAP2 concentration is also altered, but thechanges are less consistent and not as well-documented. Thesestudies are observational and do not indicate whether the reportedchanges are a cause or simply a consequence of neoplastic trans-formation. This question has been addressed in two recent publica-tions, both of which provide strong evidence to suggest thatIQGAPs contribute to tumorigenesis. In the first study, Jadeskiet al. [23] manipulated intracellular concentrations of IQGAP1 inmalignant MCF-7 cells. Specific knockdown of IQGAP1 by siRNAsignificantly reduces both serum-dependent proliferation andanchorage-independent growth in soft agar. These in vitro datastrongly suggest that IQGAP1 is required for the transformed phe-notype of MCF-7 cells, a postulate validated by in vivo analysis.Subcutaneous injection into immunocompromised mice of MCF-7cells overexpressing IQGAP1 gives rise to the formation of tumorsin 100% of mice and these tumors extensively invade skeletal mus-cle [23]. Control MCF-7 cells form tumors in 60% of animals and donot invade host tissue, whereas stable knockdown of IQGAP1 re-sults in tumors in only 20% of mice and the complete abrogationof invasion. Collectively these data strongly support the hypothesisthat IQGAP1 is an important component of breast cancer. Addi-tional work is necessary to ascertain whether IQGAP1 functionsas an oncogene and is required for neoplastic transformation ofbreast epithelial cells.
IQGAP2 is expressed predominantly in liver. Consistent with itsputative role as a tumor suppressor, targeted disruption of themurine Iqgap2 gene results in the development of HCC [47]. Theneoplasia is restricted to the liver; non-hepatic tissue exhibits nodefects. Congruent with the evidence that it is an oncogene, IQ-GAP1 is necessary for Iqgap2"/" mice to develop HCC; interbreed-ing Iqgap2"/" mice into an Iqgap1-null background significantlydecreases the incidence, size and aggressiveness of the tumors. IQ-GAP1 expression is increased !9-fold in liver, but is not altered inother organs [47]. While these findings need to be validated in hu-man HCC, they strongly suggest that deregulation of IQGAP1 andIQGAP2 may underlie the pathogenesis of this disease.
7. Perspectives
IQGAP1 is frequently overexpressed in cancer while IQGAP2expression is reduced in some neoplasms. The association of IQ-GAP1 with its binding partners Cdc42, Rac1, E-cadherin, b-catenin,components of the MAPK cascade and others may have a role intransformation and metastasis. However, the specific interactionsthat directly contribute to tumorigenesis have not been fully eluci-dated. Moreover, it is not known at which of the multiple stagesthat occur during the conversion of normal cells into malignantderivatives IQGAP1 participates. The published data, albeit limited,on IQGAP2 and IQGAP3 reveal that substantial differences existamong the IQGAP family members with respect to tissue distribu-tion, subcellular localization and binding partners. Additional workis necessary to dissect out the biological implications of these dif-ferences and determine the molecular mechanisms by which IQ-GAP2 and IQGAP3 are likely to influence tumorigenesis.
A fundamental question which remains to be answered is whattriggers overexpression of IQGAP1 and, in some neoplasms, loss ofIQGAP2? Several oncogenes and tumor suppressor genes, forexample Ras, B-Raf, p53 and APC [91,92], are mutated. Other onco-genes, like the Rho GTPases, are not mutated, but are deregulatedduring tumor progression [93]. A preponderance of evidence sug-gests that, analogous to the Rho family, the Iqgap1 gene is ampli-fied in cancer and there is little indication of IQGAP1 mutation.Genomic sequence analysis of Iqgap1 in 38 gastric cancers revealsa missense nucleotide change at an allelic frequency of only 2.6%(although other silent nucleotide changes were also observed)[94]. There are no published reports describing mutation of the Iq-gap2 or Iqgap3 genes in tumors. Further work is needed to ascertainthe molecular mechanisms by which Iqgap1 and Iqgap2 (and per-haps Iqgap3) expression is altered in neoplasia.
The cumulative evidence strongly implicates the IQGAP pro-teins in cancer. In addition to their altered levels in human neo-plasms, IQGAPs appear to contribute to tumorigenesis. Thedocumented roles for many IQGAP binding proteins in multiplestages of neoplastic transformation and metastasis, coupled withthe participation of IQGAPs in diverse signaling pathways thatare deregulated in cancer, combine to make IQGAP proteins con-ceptually appealing chemotherapeutic targets.
Acknowledgements
We apologize to all those whose primary work could not be ci-ted due to lack of space. We thank Rob Krikorian for help preparingthe manuscript and members of the Sacks laboratory, past andpresent, for insightful discussions. Work in the authors’ laboratoryis funded by the National Institutes of Health.
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Chapter 9
Regulation of MAP Kinase Signaling by Calcium
Colin D. White and David B. Sacks
Abstract
Mitogen-activated protein kinase (MAPK) signaling influences a variety of cellular responses, ranging from stimulation of cell proliferation to induction of senescence and/or apoptosis. Ca2+ is a ubiquitous intracellular signaling molecule that controls multiple processes in cells. Published evidence has identified both direct and indirect interactions between the Ca2+ and MAPK signaling pathways. Here, we describe assays to accurately determine the effect of changes in intracellular Ca2+ concentration on MAPK activation.
Mitogen-activated protein kinases (MAPKs) are ubiquitously expressed enzymes that regulate a wide variety of functions in virtually all cell types (1). The term “MAPK” usually refers to the terminal kinase in a three-tier cascade, in which MAPKs are phos-phorylated and activated by MAPK kinases (MAPKK or MEK), which themselves are phosphorylated and activated by MAPK kinase kinases (MAPKKK or MEKK). Of the major MAPK path-ways, the Ras/Raf/MEK/ERK cascade is the most widely stud-ied and is the focus of this chapter. Engagement of cell-surface receptors by extracellular signaling molecules, such as growth fac-tors, results in activation of the intracellular small G-protein Ras. The resultant change in Ras conformation facilitates its direct interaction with Raf isoforms, namely A-Raf, B-Raf, and C-Raf (also termed Raf-1) (2). The Raf proteins are serine/threonine kinases, which phosphorylate and activate MEK1 and MEK2. In turn, MEK1 and MEK2 catalyze the phosphorylation of the extracellular signal-regulated kinases, ERK1 and ERK2.
Once active, ERKs either dimerize and remain in the cytosol where they catalyze the phosphorylation of a variety of substrates, or, as monomers, translocate to the nucleus where they phospho-rylate transcription factors (3).
MAPK function is influenced by several pathways, including Ca2+ (4). For example, an increase in intracellular free Ca2+ concen-tration ([Ca2+]i) positively regulates Ras signaling in PC12 cells leading to increased ERK phosphorylation (5). Conversely, treat-ing keratinocytes with Ca2+ inhibits activation of ERK by epidermal growth factor (EGF) (6). The reasons for these discrepant data are not known, but differences between the cell types are likely to con-tribute. The ability to manipulate [Ca2+]i and accurately measure active MAPK is a welcome addition to the researchers’ toolbox. In this chapter, we describe straightforward assays for evaluating the effect of Ca2+ on growth factor-induced MAPK signaling using Western blotting and confocal immunofluorescence.
The protocol described in this chapter represents probably the most widely used methods to manipulate [Ca2+]i. A23187 is a Ca2+ ionophore that causes a rapid and sustained increase in [Ca2+]i by permitting entry into the cell of extracellular Ca2+. BAPTA-AM enters cells where it chelates intracellular Ca2+, markedly reducing [Ca2+]i. These reagents therefore allow the investigator to eluci-date the regulatory effect of Ca2+ on intracellular signaling.
In order to identify the source of the Ca2+ responsible for a specific effect, pharmacological compounds are available (which selectively modulate individual Ca2+ channels or pumps (Table 1)). Each compound can be broadly characterized as inducing either an “on” or an “off” signal (Fig. 1). On signals increase [Ca2+]i, while off signals reduce it. These are discussed in more detail below.
Certain extracellular stimuli induce a rise in [Ca2+]i. The increase in [Ca2+]i is mediated either by Ca2+ entering the cell from the outside (across the plasma membrane) or by release from intracel-lular stores. There are three classes of channels in the plasma membrane which facilitate Ca2+ influx from the outside (7). Voltage-activated Ca2+ channels respond to changes in the mem-brane potential of the cell, while ligand-activated Ca2+ channels are opened in response to the binding of a specific ligand. Store-activated Ca2+ channel opening is stimulated by the emptying of intracellular Ca2+ stores.
Ca2+ is released from the endoplasmic reticulum, an organelle that acts as an intracellular Ca2+ store. The mechanism underlying this release is similar to that of ligand-activated Ca2+ channels, but the activating ligands differ. The best studied examples are the inositol triphosphate (IP3) and ryanodine receptors, which may be modulated by binding of their cognate ligands, IP3 and ryanodine, respectively. Interestingly, the most important regulator of Ca2+
1.1. Manipulation of [Ca2+]i
1.1.1. “On” Signals
153Regulation of MAP Kinase Signaling by Calcium
Tabl
e 1
Phar
mac
olog
ical
age
nts
used
to s
elec
tivel
y m
anip
ulat
e [C
a2+] i
Site
of
man
ipul
atio
nCo
mpo
und
Effe
ct o
n [C
a2+] i
Mod
e of
act
ion
Solu
bilit
yNo
tes
Volta
ge-a
ctiv
ated
C
a2+ c
hann
els
-Aga
toxi
n!
Inhi
bits
P-t
ype
Ca2+
cha
nnel
sH
2O
(±)-
Bay
K 8
644
+A
ctiv
ates
L-t
ype
Ca2+
cha
nnel
sE
tOH
/H2O
Indu
ces p
44/4
2 M
APK
act
ivat
ion
in
Jurk
at c
ells
-Con
otox
in!
Inhi
bits
N-t
ype
Ca2+
cha
nnel
sH
2O(+
)-ci
s-Dilt
iaze
m
hydr
ochl
orid
e!
Inhi
bits
L-t
ype
Ca2+
cha
nnel
sM
eOH
/H2O
Cau
ses C
a2+ r
elea
se fr
om in
trac
ellu
lar
stor
es in
neu
trop
hils
Mib
efra
dil
dihy
droc
hlor
ide
!In
hibi
ts T
-typ
e C
a2+ c
hann
els
H2O
Inhi
bits
L-t
ype
Ca2+
cha
nnel
s at h
igh
conc
entr
atio
nsN
eom
ycin
tr
isulfa
te!
Inhi
bits
vol
tage
-act
ivat
ed C
a2+
chan
nels
H2O
No
effe
ct o
n N
a+ /C
a2+ a
ntip
orte
r in
neur
ons
Nife
dipi
ne!
L-ty
pe C
a2+ c
hann
el a
ntag
onist
DM
SO/
EtO
HIn
duce
s apo
ptos
is in
hum
an g
liobl
as-
tom
a ce
llsN
imod
ipin
e!
L-ty
pe C
a2+ c
hann
el a
ntag
onist
MeO
HPh
otos
ensit
ive
Rec
epto
r-ac
tivat
ed
Ca2+
cha
nnel
sR
uthe
nium
red
!In
hibi
ts R
yR-a
ctiv
ated
Ca2+
ch
anne
lsH
2OM
ay a
lso in
hibi
t vol
tage
-act
ivat
ed C
a2+
chan
nels
Rya
nodi
ne+/
!Lo
cks R
yR-a
ctiv
ated
Ca2+
ch
anne
ls in
a h
alf o
pen
stat
e at
nM
con
cent
ratio
ns. F
ully
cl
oses
them
in th
e µM
rang
e
DM
SO/
EtO
HR
yRs a
re e
xpre
ssed
prim
arily
in sk
elet
al
and
card
iac
mus
cle,
and
the
brai
n
Xes
tosp
ongi
n C
!In
hibi
ts I
P 3 rec
epto
r-ac
tivat
ed
Ca2+
cha
nnel
sD
MSO
/E
tOH
May
also
inhi
bit v
olta
ge-a
ctiv
ated
Ca2+
ch
anne
ls
Ca2+
-AT
Pase
sC
yclo
piaz
onic
aci
d+
Inhi
bits
ER
/SR
Ca2+
-AT
Pase
DM
SOTo
xic
at h
igh
conc
entr
atio
nsT
haps
igar
gin
!In
hibi
ts E
R/S
R C
a2+-A
TPa
seD
MSO
/E
tOH
Wid
ely
used
, pot
ent,
cell-
perm
eabl
e in
hibi
tor
+, in
crea
ses [
Ca2+
] i; !,
dec
reas
es [
Ca2+
] iSR
sarc
opla
smic
ret
icul
um; E
R e
ndop
lasm
ic r
etic
ulum
; RyR
rya
nodi
ne r
ecep
tor
154 White and Sacks
channels on intracellular stores is Ca2+ itself. This observation forms the basis of the concept of Ca2+-induced Ca2+ release (8, 9).
Off signals involve the rapid removal of intracellular free Ca2+ from the cytoplasm by a variety of pumps and exchangers. Ca2+ can be pumped out of the cell by Ca2+-ATPases or Na+/Ca2+ exchangers located on the plasma membrane. Alternatively, Ca2+ can be moved into intracellular storage compartments by Ca2+-ATPases on the endoplasmic reticulum or through Ca2+ uniporters on the inner mitochondrial membrane.
Unless otherwise stated, all reagents are stored at room temperature (~22°C).
1. Dulbecco’s Modified Eagle’s Medium (DMEM) supple-mented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/glutamine (PSG). Store at 4°C.
2. DMEM supplemented with 1% PSG and 1 mM 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Store at 4°C.
3. 0.05% Trypsin/ethylenediamine tetraacetic acid (EDTA). Store for up to 1 month at 4°C.
4. Sterile phosphate-buffered saline (PBS).
1.1.2. “Off” Signals
2. Materials
2.1. Cell Culture, Treatment, and Lysis
Fig. 1. The basic Ca2+ signaling network. A stimulus activates various “on” or “off” signals. “On” signals trigger an increase in [Ca2+]i which, in turn, induces Ca2+-mediated signaling events. “Off” signals restore [Ca2+]i to its resting level.
155Regulation of MAP Kinase Signaling by Calcium
5. A23187 (Sigma, St. Louis, MO) (50 mg/ml in dimethyl sulfoxide (DMSO)). Store in single use aliquots at !80°C. See Notes 1 and 2.
6. 1,2-Bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid (tetra(acetoxymethyl) ester) (BAPTA-AM) (Sigma, St. Louis, MO) (15 mg/ml in DMSO). Store in single use aliquots at !80°C. See Notes 3 and 4.
7. EGF (Gibco, Carlsbad, CA) (1 mg/ml in sterile PBS). Store in single use aliquots at !80°C. See Note 5.
8. PBS: 150 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.4). Store at 4°C.
9. Lysis buffer: 50 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100. Store at 4°C. Prior to use, add 1% 0.1 M phenyl-methanesulfonyl fluoride (PMSF), 0.1% Protease Inhibitor Cocktail (Sigma, St. Louis, MO), 10 µg/ml leupeptin, 1% Phosphatase Inhibitor Cocktail 1 (Sigma, St. Louis, MO) and 1% Phosphatase Inhibitor Cocktail 2 (Sigma, St. Louis, MO).
10. Disposable cell lifters. 11. 6" Sample buffer: 180 mM Tris (pH 6.8), 12% (w/v) sodium
dodecyl sulfate (SDS), 50% glycerol, 10% (w/v) dithiothre-itol, 0.006% (w/v) bromophenol blue. Store at 4°C.
3. Protogel (National Diagnostics, Atlanta, GA). 4. 10% (w/v) Ammonium persulfate (APS). 5. N,N,N ,N -tetra-methyl-ethylenediamine (TEMED). 6. Isobutanol: decant 25 ml into a 500 ml spray bottle and use
vapor. 7. Running buffer: 50 mM Tris, 0.4 M glycine, 0.1% (w/v)
SDS. 8. All Blue Precision Plus Protein Standards (Bio-Rad, Hercules,
CA). Store at !20°C.
1. Transfer buffer: 30 mM Tris, 0.25 M glycine. Prior to use, to 800 ml transfer buffer add 200 ml MeOH and 2 ml 10% (w/v) SDS.
2. Prefrozen ice container. 3. Immobilon-P Transfer Membrane (0.45 m pore) (Millipore,
Bedford, MA). 4. MeOH.
2.2. SDS-Polyacrylamide Gel Electrophoresis
2.3. Western Blotting for Active MAPK
156 White and Sacks
5. Tris-buffered saline with Tween (TBS-T): 10 mM Tris, 150 mM NaCl, 0.2% Tween-20 (pH 8.0).
6. Blocking buffer: 4% (w/v) bovine serum albumin (BSA) in TBS-T. Store at 4°C.
monoclonal antibody (Cell Signaling Technology, Danvers, MA (catalog no. 4370)). Store at !20°C.
9. Secondary antibody: Horseradish peroxidase (HRP)-linked anti-rabbit immunoglobulin G (GE Healthcare, Bucking-hamshire, UK). Store at 4°C.
10. Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, Bedford, MA). Store at 4°C.
11. Kodak BioMax XAR X-ray Film (Carestream Health, Rochester, NY).
1. Stripping buffer: 62.5 mM Tris–HCl (pH 6.8), 2% (w/v) SDS, 0.7% -mercaptoethanol. Make fresh as required.
2. Primary antibody: Anti-p44/42 MAPK mouse monoclonal antibody (Cell Signaling Technology, Danvers, MA (catalog no. 4696)). Store at !20°C.
3. Secondary antibody: HRP-linked anti-mouse immunoglobu-lin G (GE Healthcare, Buckinghamshire, UK). Store at 4°C.
4. Immobilon Western Chemiluminescent HRP Substrate Kit. Store at 4°C.
5. Kodak BioMax XAR X-ray Film.
1. Microscope Cover Glass. 2. Lab-Tek four-well Glass Chamber Slides. 3. PBS. Store at 4°C. 4. 4% (w/v) Paraformaldehyde (PFA) in PBS. Store at 4°C. See
BSA in PBS. Store at 4°C. 6. Antibody diluent: 0.2% Triton X-100, 1% BSA in PBS. Store
at 4°C. 7. Primary antibodies: Anti-phospho-p44/42 MAPK rabbit
monoclonal antibody and anti-p44/42 MAPK mouse mono-clonal antibody. Store at !20°C.
8. Secondary antibodies: Alexa-Fluor 488-labeled anti-rabbit immunoglobulin G (Molecular Probes, Carlsbad, CA) and Alexa-Fluor 488-labeled anti-mouse immunoglobulin G (Molecular Probes, Carlsbad, CA). Store both in light-protected single use aliquots at !20°C.
2.4. Stripping Blots and Reprobing for Total MAPK
2.5. Confocal Immunofluorescence for Active and Total MAPK
157Regulation of MAP Kinase Signaling by Calcium
9. Nuclear stain: 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Carlsbad, CA). Store in light-protected single use aliquots at !20°C. See Notes 7 and 8.
10. Mounting medium: PermaFluor Aqueous Mounting Medium (Fisher, Pittsburgh, PA). Store at 4°C.
1. Passage cells when approaching confluence by washing with sterile PBS and detaching with prewarmed 0.05% trypsin/EDTA. One 100 mm dish is required for each data point (each dish holds a volume of ~5–10 ml). Allow cells to attach and approach confluence in prewarmed DMEM supple-mented with 10% FBS and 1% PSG.
2. At 90–100% confluence, rinse cells twice with sterile PBS. Starve cells of serum by incubating in prewarmed (37°C) DMEM supplemented with 1% PSG and 1 mM HEPES for 16 h at 37°C.
3. Prepare all materials for cell treatment and lysis (see Notes 1–5). Other materials also required at this stage include three prechilled and labeled microcentrifuge tubes per dish, PBS and lysis buffer (both at 4°C), disposable cell lifters, 70% EtOH and 6" sample buffer.
4. Aspirate growth medium from each 100 mm dish and replace with medium containing either vehicle (DMSO), A23187 or BAPTA-AM as appropriate. Incubate for 20 min at 37°C. See Notes 1–4 and 9.
5. Treat each experimental culture with either vehicle (0.01% BSA) or 100 ng/ml EGF as appropriate. Incubate for 5 min at 37°C. See Note 10.
6. Immediately place all 100 mm dishes on ice and aspirate growth medium. Wash rapidly with cold PBS. Aspirate and add 500 l cold lysis buffer to each dish. Swirl dishes gently to ensure even coverage. See Note 11.
7. Using a disposable cell lifter, scrape the contents of each 100 mm dish into an appropriately labeled prechilled micro-centrifuge tube. Rinse the disposable cell lifter in 70% EtOH between samples. Sonicate twice at high power for 5–10 s and clarify by high speed centrifugation (~15,000 " g) for 10 min at 4°C. See Note 12.
8. Carefully aspirate supernatant and transfer into a separate appropriately labeled prechilled microcentrifuge tube. Discard pellet. If desired, protein concentration in an aliquot of the supernatant may be measured using the Modified Bradford Assay (Bio-Rad, Hercules, CA).
3. Methods
3.1. Cell Culture, Treatment, and Lysis
158 White and Sacks
9. To 10 l 6! sample buffer, add 40 l lysate (or an appropriate normalized volume). Mix well and boil at 100°C for 5 min. Centrifuge briefly, cool to 22°C and proceed to Sub-heading 3.2. See Notes 13–15.
This protocol describes the use of the Bio-Rad Mini-Blot Gel System (Bio-Rad, Hercules, CA). Nevertheless, it is easily adapt-able to other formats.
1. Prior to (and following) each use, clean each glass plate with 70% EtOH and rinse well with ddH2O.
2. Prepare a 1.5 mm thick separating gel of the appropriate per-centage (Table 2). After addition of TEMED, proceed imme-diately to step 3.
3. Pour the gel ensuring that ~1.5 cm of space is left at the top for the stacking gel. Use isobutanol vapor to remove any air bubbles. Polymerization should take place in 30–45 min.
4. Prepare the stacking gel by mixing 330 l Protogel, 630 l 4! Protogel Stacking Buffer, 1.53 ml ddH2O, 12.5 l 10% (w/v) APS and 2.5 l TEMED. Pour the stack, use isobutanol vapor to remove any air bubbles and carefully insert the comb. Polymerization should take place in 30–45 min. See Note 16.
5. Once the stacking gel has set, carefully remove the comb and use a 5 ml syringe fitted with a 22-gauge needle to wash the wells with running buffer.
6. Assemble the gel unit and fill each chamber with running buf-fer. Load 10 l All Blue Precision Plus Protein Standard in well 1. Each sample should be added carefully to a separate well.
3.2. SDS-PAGE
Table 2 Separating gel components for different % acrylamide gels
Typical protein size resolved (kDa)a 60–300 40–300 20–300 20–200 10–150aThe range of proteins resolved using different % gels is based on our experience using the reagents in this protocol. The use of other reagents may substantially alter these values
159Regulation of MAP Kinase Signaling by Calcium
7. Complete the assembly of the gel unit and connect to a power supply. Run at 50 mA for ~60 min or until the dye front reaches the bottom of the gel.
1. At this stage it is necessary to prepare for gel transfer. This protocol assumes the use of a “wet” transfer system but is eas-ily adaptable to the “semi-dry” equivalent. Cut a piece of Immobilon-P Transfer Membrane approximately 7 cm ! 5 cm in size and soak thoroughly for ~5 min in MeOH. After soak-ing, rinse thoroughly with ddH2O and soak in transfer buffer until SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis) is complete. Both foam pads of the transfer cassette should also be soaked thoroughly in transfer buffer for at least 30 min prior to use (see Note 17).
2. Disconnect the gel unit from the power supply and disas-semble. Using a clean razor blade, cut away the stacking gel and discard. Similarly, if still present, cut away anything below the dye front on the separating gel. Carefully submerge the remaining separating gel in transfer buffer.
3. Assemble the transfer cassette as follows: open the cassette and place one soaked foam pad on each side. Place the sepa-rating gel on a foam pad and carefully lay the Immobilon-P Transfer Membrane on top. Gently remove any air bubbles in the stack by rolling with a clean test tube, then place the other foam pad on top. Gently remove any air bubbles again and close the transfer cassette (see Note 18).
4. Place the transfer cassette into the transfer tank such that the separating gel is closest to the negative cathode and the Immobilon-P Transfer Membrane to the positive anode. This orientation is critical or the proteins will be lost. Fill the trans-fer tank with transfer buffer and drop in a small magnetic stir bar. Slot a prefrozen ice container into place.
5. Put the lid on the transfer tank and connect to a power sup-ply. Place the apparatus on a magnetic stirrer and switch on. Transfer at 100 V for 1 h (see Notes 19 and 20).
6. Disconnect the transfer tank from the power supply and remove the transfer cassette. Discard the separating gel and place the Immobilon-P Transfer Membrane in a clean plastic container. If the transfer was successful, the All Blue Precision Plus Protein Standards should be clearly visible.
7. Incubate the Immobilon-P Transfer Membrane in 10 ml blocking buffer for 1 h at 22°C or overnight at 4°C.
8. Prepare the primary antibody solution as follows: To 10 ml blocking buffer add 100 l 10% sodium azide and 10 l anti-phospho-p44/42 MAPK. Store at 4°C.
3.3. Western Blotting for Active MAPK
160 White and Sacks
9. Remove the blocking buffer and incubate the Immobilon-P Transfer Membrane in 10 ml primary antibody solution for 1 h at 22°C or overnight at 4°C (see Note 21).
10. Remove the primary antibody solution and wash the Immobilon-P Transfer Membrane three times for 10 min each with 20 ml TBS-T.
11. The secondary antibody is freshly prepared for each experi-ment. To 10 ml blocking buffer add 2 l HRP-linked anti-rabbit immunoglobulin G. After washing is complete, add the secondary antibody and incubate for 1 h at 22°C.
12. Remove the secondary antibody and wash the Immobilon-P Transfer Membrane three times for 10 min each with 20 ml TBS-T.
13. Once the final wash is finished, mix together 1 ml of each reagent in the Immobilon Western Chemiluminescent HRP Substrate Kit and pour on to the Immobilon-P Transfer Membrane. Rotate using forceps for 1.5 min to ensure even coverage and place between the leaves of a lightweight sheet protector that has been pre-cut to the same size as an X-ray film cassette.
14. Place the sheet protector in the X-ray film cassette and pro-ceed immediately to a dark room. Delays at this stage in the protocol will result in loss of the chemiluminescent signal.
15. Under safe light conditions, place a sheet of Kodak BioMax XAR X-ray film into the cassette and expose for a suitable time. For most proteins, including phospho-p44/42 MAPK, typical exposure times range between 1 s and 1 min.
1. Upon satisfactory exposure of active phosphorylated MAPK, it is necessary to strip the Immobilon-P Transfer Membrane and reprobe with an antibody that recognizes both phosphory-lated and nonphosphorylated MAPK. This provides a loading control and allows quantification of the various EGF-stimulated responses (see Note 22).
2. Using a preheated waterbath, incubate the Immobilon-P Transfer Membrane in 50 ml stripping buffer for 30 min at 55°C (see Note 23).
3. Remove the stripping buffer and wash the Immobilon-P Transfer Membrane six times for 5 min each with 20 ml TBS-T.
4. Incubate the Immobilon-P Transfer Membrane in 10 ml blocking buffer for 1 h at 22°C or overnight at 4°C.
5. Prepare the primary antibody solution as follows: To 10 ml blocking buffer add 100 l 10% sodium azide and 10 l anti-p44/42 MAPK. Store at 4°C.
3.4. Stripping Blots and Reprobing for Total MAPK
161Regulation of MAP Kinase Signaling by Calcium
6. Remove the blocking buffer and incubate the Immobilon-P Transfer Membrane in 10 ml primary antibody solution for 1 h at 22°C or overnight at 4°C (see Note 21).
7. Remove the primary antibody solution and wash the Immobilon-P Transfer Membrane three times for 10 min each with 20 ml TBS-T.
8. As before, the secondary antibody is freshly prepared for each experiment. To 10 ml blocking buffer add 2 l HRP-linked anti-mouse immunoglobulin G. After washing is complete, add the secondary antibody and incubate for 1 h at 22°C.
9. Remove the secondary antibody and wash the Immobilon-P Transfer Membrane three times for 10 min each with 20 ml TBS-T.
10. Repeat steps 13–15 in Subheading 3.3. Typical exposure times for p44/42 MAPK range from between 1 and 20 s.
1. Passage cells when approaching confluence by washing with sterile PBS and detaching with prewarmed 0.05% trypsin/EDTA. One well of a Lab-Tek 4-well Glass Chamber Slide is required for each data point (each well holds a volume of ~500 l). Allow cells to attach and approach confluence in pre-warmed DMEM supplemented with 10% FBS and 1% PSG.
2. At 70–80% confluence, rinse cells twice with sterile PBS. Starve cells of serum by incubating in prewarmed (37°C) DMEM supplemented with 1% PSG and 1 mM HEPES for 16 h at 37°C (see Note 24).
3. Prepare all materials for cell treatment and permeabilization (see Notes 1–5). Other materials also required at this stage include PBS, PFA, and blocking and permeabilization buffer (all at 4°C).
4. Aspirate growth medium from each well and replace with medium containing either vehicle (DMSO), A23187 or BAPTA-AM as appropriate. Incubate for 20 min at 37°C (see Notes 1–4 and 9).
5. Treat each experimental culture with either vehicle (0.01% BSA) or 100 ng/ml EGF as appropriate. Incubate for 5 min at 37°C (see Note 10).
6. Immediately place all Lab-Tek four-well Glass Chamber Slides on ice and aspirate growth medium. Wash rapidly with cold PBS. Aspirate and add 500 l cold PFA to each well. Leave for 20 min at 22°C.
7. Wash twice with cold PBS. Aspirate and add 500 l cold blocking and permeabilization buffer to each well. Leave for 1 h at 22°C.
3.5. Confocal Immunofluorescence for Active and Total MAPK
162 White and Sacks
8. The primary antibody solution is prepared freshly for each experiment. To 500 l antibody diluent add 5 l anti-phospho-p44/42 MAPK or 5 l anti-p44/42 MAPK.
9. Remove the blocking and permeabilization buffer and incu-bate the experimental cultures in 500 l primary antibody solution overnight at 4°C.
10. Remove the primary antibody solution and wash three times with cold PBS. The experimental cultures are protected from light for all subsequent steps.
11. The secondary antibody is freshly prepared for each experi-ment. To 500 l antibody diluent add 1 l Alexa-Fluor 488-labeled anti-rabbit immunoglobulin G or 1 l Alexa-Fluor 488-labeled anti-mouse immunoglobulin G. Add the secondary antibody and incubate for 1 h at 22°C.
12. Remove the secondary antibody and wash three times with cold PBS. Incubate the experimental cultures in 500 l DAPI for 5 min at 22°C (see Notes 7 and 8).
13. Remove the DAPI and wash three times with cold PBS. Aspirate all the liquid and carefully remove the wells using the supplied tool. Apply ~2–3 ml PermaFluor Aqueous Mounting Medium and a Microscope Cover Glass. Leave in light-protected conditions for 24 h at 4°C.
14. View the slides using phase-contrast microscopy to locate the cells and identify the focal plane. Under confocal conditions, excitation at 488 nm induces green fluorescence for either phospho-p44/42 MAPK or p44/42 MAPK. Excitation at 364 nm induces blue fluorescence for DAPI (see Note 25).
1. Working solutions of A23187 are prepared by diluting to 50 g/ml in DMSO and subsequent dilution to 5 ng/ml in DMEM supplemented with 1% PSG and 1 mM HEPES.
2. A23187 is a selective Ca2+ ionophore (10). It greatly increases the ability of divalent ions to cross biological membranes by forming stable 2:1 complexes with them, thus rendering them cell-permeable. A23187 is commonly used to increase [Ca2+]i in intact cells. A less Ca2+-selective alternative is Ionomycin (Sigma, St. Louis, MO).
3. Working solutions of BAPTA-AM are prepared by diluting to 30 g/ml in DMSO and subsequent dilution to 30 ng/ml in DMEM supplemented with 1% PSG and 1 mM HEPES.
4. Notes
163Regulation of MAP Kinase Signaling by Calcium
4. BAPTA-AM is a Ca2+ chelator with 105-fold greater affinity for Ca2+ than for Mg2+ (10). Once inside the cell, the ace-toxymethyl moiety is hydrolyzed by cytosolic esterases and BAPTA, which is unable to cross the plasma membrane, is trapped intracellularly.
5. Working solutions of EGF are prepared by diluting to 100 g/ml in 0.01% BSA.
6. To dissolve PFA, heat to ~50°C with constant stirring. Precipitation after long term storage indicates that the solu-tion should be discarded.
7. Working solutions of DAPI are prepared by dilution to 200 ng/ml in PBS.
8. DAPI is a known carcinogen. Always wear gloves. 9. It is our experience that the concentrations and incubation
times of A23187 and BAPTA-AM we have suggested are suf-ficient to elicit effects on EGF-induced MAPK activation. Nevertheless, incubation of each reagent at different concen-trations for different times should be performed in order to optimize the protocol for each cell type.
10. EGF typically induces maximal p44/42 MAPK activation ~2–5 min poststimulation. Nevertheless, stimulation at dif-ferent concentrations for different times should be performed to optimize the protocol for each cell type.
11. 500 l is the recommended initial lysis volume. It can be decreased in order to concentrate protein should a satisfac-tory phospho-p44/42 MAPK signal not be obtained.
12. Ear protection should be worn when using a sonicator. 13. 6! Sample buffer should be warmed to 22°C before use to
allow accurate pipetting. 14. Microcentrifuge tube caps should be “locked” shut in order
to prevent them springing open during boiling which may result in loss of some of the sample. If using conventional 1.5 ml microcentrifuge tubes, Microtube Lid Locks (Fisher, Pittsburgh, PA) provide an inexpensive way to achieve this.
15. If required, the protocol may be stopped at this point and the samples stored at "80°C.
16. We use 1.5 mm thick 10-well combs. Both 12- and 15-well models are also available, but limit the sample volume that may be loaded in each well to ~30 l and ~10 l, respectively.
17. The Immobilon-P Transfer Membrane is extremely hydro-phobic and will not wet in aqueous solutions unless prewet in methanol. After prewetting, do not let the membrane dry. In the event it does dry, it should again be wet in MeOH.
164 White and Sacks
18. Air bubbles should be carefully rolled out to avoid disturbing the flow of current from the negative cathode to the positive anode and thus the transfer of proteins from the separating gel to the Immobilon-P Transfer Membrane.
19. Stir at low speed to avoid the introduction of air bubbles. 20. Coomassie Blue staining can be used to evaluate transfer effi-
ciency. After gel transfer, remove the Immobilon-P Transfer Membrane and incubate the separating gel in Coomassie Blue stain (50% MeOH, 10% acetic acid, 40% ddH2O, 0.2% (w/v) Coomassie Blue) for 1 h at 22°C. After staining, wash with ddH2O and incubate in Gel Destain Buffer (10% MeOH, 10% acetic acid, 80% ddH2O) for ~16 h at 22°C.
21. It is our experience that both anti-phospho-p44/42 MAPK and anti-p44/42 MAPK may be reused ~20 times after which fresh primary antibody solutions should be prepared.
22. Densitometry should be performed to quantify the effect of EGF on MAPK activation. Scan the exposed Kodak BioMax XAR X-ray film into a computer and analyze using a suitable quantification program. Several software packages are avail-able. We recommend ImageJ (available free from http://rsb.info.nih.gov/ij/index.html) as it is both accurate and easy to use. The densitometrical value of each sample when probed with anti-phospho-p44/42 MAPK should be corrected for the value of the same sample when probed with anti-p44/42 MAPK.
23. Incubation of the Immobilon-P Transfer Membrane in strip-ping buffer may not remove all of the protein-bound primary antibody. When reprobing for a protein of size similar to that already imaged, it is advisable to verify that all of the primary antibody has been removed. After completing step 4 in Subheading 3.4, add the secondary antibody and incubate for 1 h at 22°C without first adding the primary antibody solu-tion. Remove the secondary antibody and wash the Immobilon-P Transfer Membrane three times for 10 min each with 20 ml TBS-T. Repeat steps 13–15 in Subheading 3.3. A positive signal indicates that not all of the primary antibody has been removed during the stripping process. Stripping again, or increasing the temperature at which the Immobilon-P Transfer Membrane is incubated in stripping buffer to 80°C, may solve this problem.
24. Seventy to eighty percent confluence is recommended for microscopy studies in order to ensure that individual cells are clearly visible under the microscope.
25. Confocal laser scanning microscopy allows high-resolution optical images to be obtained. The defining feature is the abil-ity to optically section a sample and thus effectively produce a
three-dimensional image. Confocal microscopes are commonly used in immunofluorescence studies as they generally obtain much higher quality images than would be afforded by a fluo-rescent microscope.
Acknowledgments
We thank Zhigang Li for critically reviewing the text prior to sub-mission and other members of the Sacks laboratory, past and present, for insightful discussions. Work in the authors’ labora-tory is funded by the National Institutes of Health (to D.B.S) and the Department of Defense Breast Cancer Research Program (to C.D.W).
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8/25/10 11:30 AMIQGAP1 is a Novel HER2 Binding Partner and Regulates HER2-Mediated Cell Proliferation -- White et al. 24 (1): 421.10 -- The FASEB Journal
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Year: Vol: 24 Page:
421.10
IQGAP1 is a Novel HER2 Binding Partnerand Regulates HER2-Mediated CellProliferation
Colin David White1, Zhigang Li1, Mario Niepel2 andDavid Barry Sacks1
1 Department of Pathology, Brigham and Women’s Hospital and Harvard MedicalSchool, Boston, MA2 Department of Systems Biology, Harvard Medical School, Boston, MA
ABSTRACT
The receptor tyrosine kinase HER2 is overexpressed in 25–30% of breast carcinomas. These tumors oftenhave an increased rate of proliferation and give rise to more frequent metastases than HER2(–) neoplasms.HER2(+) breast cancer is treated with Trastuzumab, but ~60% of patients do not respond. Of those who do,~15% subsequently develop metastatic disease. An understanding of the molecular mechanisms of HER2signaling is necessary to develop novel therapeutics. IQGAP1 is a ubiquitously expressed 190 kDa scaffoldprotein. Through interaction with multiple binding partners, IQGAP1 regulates processes such as cellproliferation and migration, cell-cell adhesion and cytoskeletal remodeling. IQGAP1 plays defined roles inseveral human malignancies, including those of the breast. Here, we investigate the interaction betweenIQGAP1 and HER2. In vitro assays using purified IQGAP1 and the GST-tagged intracellular domain ofHER2 (GST-HER2) revealed that IQGAP1 binds HER2. GST-HER2 binds IQGAP1 in Sk-BR-3 cells, andendogenous HER2 co-immunoprecipitates with endogenous IQGAP1 from cell lysates. Analogous to otherIQGAP1 binding partners, calmodulin abrogates the association of IQGAP1 with HER2. Overexpression ofIQGAP1 significantly increases Sk-BR-3 cell proliferation, while siRNA-mediated knockdown of IQGAP1significantly reduces proliferation. These data suggest that IQGAP1 may be a potential target for HER2(+)breast cancer therapy.
Work in the authors’ laboratory is funded by the NIH (to D.B.S.) and the DoD BCRP (to C.D.W).
September 22, 2010 Progress Review Committee Congressionally-Directed Medical Research Program: Re: BC087504 - The Role of IQGAP1 in Breast Carcinoma Dear Committee Members: I am writing this letter of support for Dr. Colin White, the named PI on BCRP grant number BC087504 - The Role of IQGAP1 in Breast Carcinoma. Dr. White encountered considerable problems during year 1 of this award with the studies proposed. Stable cell lines were necessary to perform most of the planned experiments. Unfortunately, Dr. White was plagued with repeated infections of his cell cultures. The problem was caused by others who use the shared cell culture facilities made available to him within the Department of Pathology, Brigham and Women’s Hospital. Although we attempted several strategies to overcome these issues, none of the measures we employed was successful. As a result, the Statement of Work for the remainder of this award has been changed. Please note, the overall boundaries of the original proposal remain the same. Dr. Katherine Moore, grants manager at the CDMRP, has approved this revision. In my opinion Dr. White has made very satisfactory progress with the revised project. He has a good work ethic and has been very productive during his time in my laboratory. I have no hesitation in recommending that his funding continue through years 2 and 3. Yours sincerely,
David B. Sacks, M.B., Ch.B., FRCPath
~ BRIGHAM AND WOMEN'S HOSPITAL
Department of Pathology Breast Pathology Services 75 Francis Street, TH613 Boston, Massachusetts 02115
Deborah A. Dillon, M.D. Assistant Professor of Pathology
I am writing to indicate my enthusiastic support for your proposed work "The Role of IOGAP1 in Breast Carcinoma". Your recent preliminary results implying a possible role for IOGAP1 in HER2 signaling are exciting and clearly merit further investigation. As you know, a significant number of patients whose breast tumors are HER2 positive fail initially to respond to trastuzumab. Furthermore, of those who do initially respond , many eventually develop resistance. Thus, there is a pressing need for the discovery and validation of potential alternative targets in this pathway.
As a breast pathologist at Brigham and Women's Hospital/Dana Farber Cancer Institute with a particular interest in the development of novel markers of potential diagnostic/therapeutic relevance for breast cancer patients, I have considerable experience in the molecular and immunohistochemical analysis of formalin-fixed paraffin embedded breast cancer tissues. Tissues that we may access for the purposes of the proposed study include anonymous excess tumor tissue of patients undergoing resection at Brigham and Women's Hospital. In addition, I have recently assembled into tissue microarrays the tissue blocks of >600 patients consented for linkage of molecular markers with treatment and outcome. Of particular importance to this proposal, both the anonymous and the linked tissues are of known estrogen receptor, progesterone receptor and HER2 status, allowing us to determine if IOGAP1 expression might be linked to one of the important clinical treatment tumor phenotypes.
In summary, I can provide you with the tissues you need to accomplish the goals of the proposed research and look forward to assisting in the analysis of IOGAP1 in these tissues. This is an exciting project with the potential to broaden the number of specific molecular targets available for breast cancer therapy.
Best regards ,
Deborah Dillon, MD Assistant Professor of Pathology Breast Pathologist, Brigham and Women's Hospital/Dana-Farber Cancer Institute
